The long-term goal of this proposal is to provide the training and support for the applicant to become an independent physician-scientist with a career focused on studying the genomics and epigenomics of acute leukemia. The goals for the applicant include: obtaining practical and didactic training in scientific methodology, refining technical skills, and publishing the outlined research in top-tier scientific journals. The long-tem goal of the research is to define the role of KDM6A in normal and leukemic hematopoiesis. KDM6A encodes a H3K27 histone demethylase on the X chromosome that is mutated in a wide range of human cancers, including acute myeloid leukemia. Inactivating mutations of Kdm6a are the most common acquired progression events in our mouse model of acute promyelocytic leukemia (APL). To define the functional consequences of Kdm6a inactivation, we propose the following aims: Specific Aim 1: We will analyze a Kdm6a conditional knockout mouse to define the role of Kdm6a in normal and leukemic hematopoiesis. We will cross Kdm6a conditional knockout mice (with LoxP sites flanking the 3rd exon) with Vav-Cre transgenic mice to selectively delete Kdm6a in the hematopoietic lineage. We will characterize normal hematopoiesis using competitive repopulation assays, flow-based analysis of hematopoietic progenitors, and serial replating experiments. For these experiments, we will analyze 6-8 week old male Vav-Cre mice hemizygous for the floxed Kdm6a allele, along with the appropriate control mice. We will establish a tumor watch to determine if Kdm6a inactivation is associated with spontaneous leukemia development or bone marrow failure. We will intercross Kdm6a conditional knockout mice (with the Vav-Cre allele) with Ctsg-PML-RARA mice to define changes in cell growth, differentiation, and self renewal, and we will determine whether Kdm6a deficiency alters APL latency, penetrance, or phenotype in a tumor watch study. Specific Aim 2: We will define the epigenetic consequences of Kdm6a inactivation using RNA-Seq and ChIP-Seq. We will perform RNA-Seq on mouse APL tumors with and without spontaneous Kdm6a deletions or mutations, and on RNA derived from bone marrow cells from young, male Vav-Cre mice hemizygous for the Kdm6a conditional allele, along with the appropriate control animals. We will perform ChIP-Seq on mouse APL tumors with and without spontaneous Kdm6a deletions or mutations. We will use an anti-H3(tri-methyl K27) antibody to determine if changes in H3K27 methylation occur, and also with an anti-H3(tri-methyl K4) antibody, since Kdm6a inactivation may alter H3K4 methylation. Finally, we will perform RNA-Seq and ChIP-Seq on selected cell populations and leukemias (if they develop) in the male Vav-Cre mice hemizygous for the Kdm6a conditional allele, and APL tumors that arise in Ctsg-PML-RARA mice with and without the engineered deletion of Kdm6a. We will therefore comprehensively identify and analyze the epigenetic and transcriptional alterations associated with Kdm6a inactivation in wild type vs. PML-RARA expressing hematopoietic and leukemia cells.